Erythropoietin treatment for anemia and hematopoietic/erythroid progenitor cell responses

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Erythropoietin is required for red blood cell production and a role for nitric oxide to increase erythropoiesis is suggested in animal models of chronic stress and in hydroxycarbamide induction of human fetal hemoglobin. We previously observed that erythropoietin protective activity in select non-erythroid tissue was mediated in part by nitric oxide synthase production of nitric oxide. Erythropoietin neuro-protection and cardio-protection in animal models required activity of neuronal nitric oxide synthase and endothelial nitric oxide synthase, respectively. We used in vivo mouse models of erythropoietin stimulated erythropoiesis and ex vivo primary erythroid progenitor cell cultures to demonstrate the differential role of endothelial nitric oxide synthase and neuronal nitric oxide synthase in erythropoietic response to erythropoietin treatment. Hematocrit level in endothelial nitric oxide synthase knockout mice were comparable to wild type mice, and erythropoietin treatment resulted in comparable erythropoietic response and increase in hematocrit. In contrast, mice that lack neuronal nitric oxide synthase exhibit a blunted erythropoietic response to erythropoietin administration resulting in a smaller increase in hematocrit. Erythroid colony assays of cultured bone marrow cells from endothelial nitric oxide synthase knockout mice, neuronal nitric oxide synthase knockout mice and wild type mice yielded comparable numbers of colonies at low erythropoietin concentration. At high erythropoietin concentration, colony numbers and size significantly increased in bone marrow cultures from wild type and endothelial nitric oxide synthase knockout mice. However, erythroid colonies in bone marrow cultures from neuronal nitric oxide synthase knockout mice remained small and fewer in number at high erythropoietin concentration. These erythroid culture studies provided evidence of an intrinsic defect in hematopoietic stem cells from neuronal nitric oxide synthase knockout mice that compromises the erythropoietic response when exposed to high erythropoietin concentration. Mouse transplantation of hematopoietic stem cell containing bone marrow into wild type recipient mice was used to compare the response of hematopoietic stem cells from wild type mice and from neuronal nitric oxide synthase knockout mice stimulated with high dose erythropoietin treatment in vivo. Bone marrow harvested from either neuronal nitric oxide synthase knockout donor mice or from wild type donor mice was transplanted into wild type recipient mice with busulfan conditioning. The level of engraftment achieved was comparable in transplants from neuronal nitric oxide synthase knockout mice and from wild type mice. Erythropoietin treatment increased hematocrit in all transplanted mice. However, mice transplanted with wild type bone marrow reached a higher hematocrit than mice transplanted with neuronal nitric oxide synthase knockout mice bone marrow. The decreased erythropoietic response with erythropoietin treatment in mice transplanted with neuronal nitric oxide synthase knockout mice bone marrow supports the hypothesis that neuronal nitric oxide synthase activity in hematopoietic/erythropoietic progenitor cells contributes directly to erythropoietin stimulated erythropoiesis and expansion of the erythroid lineage during erythroid differentiation, and is required for normal erythropoietic response. Cultures of human peripheral blood CD34+ hematopoietic cells and other erythropoietin dependent erythroid cell lines showed induction of neuronal nitric oxide synthase expression during erythropoietin stimulated erythroid differentiation. Furthermore, modification of neuronal nitric oxide synthase activity adversely affected proliferation and expansion of the erythroid lineage in erythropoietin stimulated cultures of primary human peripheral blood hematopoietic progenitor cells. In the bone marrow, erythropoietin receptor is expressed in hematopoietic and non-hematopoietic cells including bone marrow stromal cells. In contrast to the protective activity of erythropoietin in select animal models of brain, heart and skeletal muscle injury, erythropoietin adversely affected bone health. Erythropoietin stimulated erythropoiesis in mice is accompanied by bone loss mediated by non-erythroid erythropoietin receptor expression in bone marrow stromal cells, especially osteoblasts. An in vivo bone formation assay using transplantation of bone marrow stromal cells absorbed onto a collagen scaffold demonstrated that that high dose erythropoietin directly affects bone marrow stromal cell adipogenesis and osteogenesis and compromises the ability to form bone ossicles. Analysis of U.S. Renal Data System (USRDS) datasets revealed a previously unrecognized adverse impact of EPO dose on bone fracture risk in hemodialysis patients, adding support to minimize erythropoietin dose in anemia treatment. Full knowledge of the erythropoietic and non-erythroid activities of erythropoietin and potential interacting pathways will be informative to increase awareness of possible off-target effects associated with high dose erythropoietin and for development of erythropoietin derivatives or combination therapy for specific erythroid or non-erythroid erythropoietin response.